Session 10: The Immune Response in Health and Disease
Transcript of Part 3: The Troublesome Tubercle in Tuberculosis
00:00:07.21 My name is Lalita Ramakrishnan. 00:00:09.16 I'm a professor of Immunology and Infectious diseases at the University of Cambridge. 00:00:14.08 And, in this lecture, I'm going to continue to tell you a little bit more about tuberculosis, 00:00:20.16 focusing on a structure called the tubercle. 00:00:24.13 Now, just to recapitulate the lifestyle or the life cycle of Mycobacterium tuberculosis, 00:00:33.02 which is the causative agent of tuberculosis, the bacterium that causes it, the bacteria 00:00:39.01 are exhaled by... in coughs by people who are infected, get inhaled by individuals near 00:00:48.22 them, and then they enter cells that are called macrophages. 00:00:52.18 But what happens next is that they... they induce these macrophages to form structures 00:01:01.16 called granulomas, and these granulomas can become quite elaborate. 00:01:09.14 At first, they're comprised just of macrophages, but then many other immune cells come in and 00:01:15.13 they can form of a fairly complex structure. 00:01:19.21 And another thing to note is that the macrophages within this granuloma undergo a specialized 00:01:27.15 differentiation called epithelioid transformation, where they form these finger-like, interdigitated 00:01:34.03 projections between each other to form a very compact, organized structure. 00:01:42.23 Now, this structure... pathologists call these structures granulomas, and granulomas were... 00:01:51.22 are... are associated with many, many, many diseases, both infectious and non-infectious. 00:01:59.19 And they can often be quite pathological. 00:02:02.03 But granulomas... but... but the single biggest cause of granulomas is tuberculosis. 00:02:09.13 And, in fact, granulomas were first discovered in the context of tuberculosis in 1679, some 00:02:18.13 200 years before the bacterium Mycobacterium tuberculosis was... was discovered. 00:02:25.09 And, in fact, the microbe... the... the structure used to be called a tubercle, and obviously 00:02:31.23 that... both the bacterium and the disease are named for the granuloma. 00:02:35.16 So... but it turns out that granulomas are very primitive structures; they've probably 00:02:43.03 evolved... there... they're present even in lower vertebrates in a... in a more primitive 00:02:47.11 form, and they probably evolved to wall off foreign bodies -- you could imagine a thorn 00:02:54.02 being... being circumscribed by that... by such a structure until it's... 00:02:59.03 it's sort of dissolved. 00:03:01.00 But these so-called foreign body... body granulomas have a very low turnover of macrophages; the 00:03:07.14 macrophages just come and sit there and it's not a particularly inflammatory structure, 00:03:11.14 or so it's thought. 00:03:13.19 In contrast, the types of granulomas that form with tuberculosis, and pretty much any 00:03:18.02 medically significant granuloma, tend to be high turnover granulomas where there's a rapid 00:03:25.10 death of macrophages and a repopulation by new ones, 00:03:28.22 and they can be very inflammatory structures. 00:03:31.22 There are many traditional animal models that are used to study TB. 00:03:36.11 The oldest ones are the ones... the rabbit and the guinea pig, which were used by 00:03:42.19 Villemin and Koch, respectively, at the time that they discovered TB. 00:03:48.11 They used these animals to... to pass the bacterium from one animal to another to show 00:03:54.19 that it was associated with... with TB. 00:03:58.18 The most commonly used model now is the... is the mouse. 00:04:01.21 And this makes a lot of sense because mice have a wonderful array of 00:04:08.03 immunological and genetic tools. 00:04:10.06 One issue is that mice don't... that most mice... mouse strains don't develop the... 00:04:18.07 the nice, tight granulomas that are associated with human disease, but there are some recently 00:04:25.06 identified mouse strains that do, and so those could actually be quite good. 00:04:33.07 Another more recently used model is the non-human primate, and this is a good model because 00:04:40.04 it really recapitulates human disease. 00:04:43.03 But the problem is, of course, they're expensive, there are ethical considerations, and obviously 00:04:48.06 cannot be used widely. 00:04:50.19 So, in... on this backdrop, my story and my engagement with TB and the granuloma came 00:05:00.24 when, as a postdoctoral fellow at UCSF -- I was a clinical infectious disease fellow and 00:05:07.08 had to do a postdoctoral fellowship -- I approached Stanley Falkow at Stanford to... to go to 00:05:14.07 his lab and study TB, which he didn't study at the time, 00:05:17.14 but he studied many other bacterial pathogens. 00:05:20.22 And Stanley said to me, forget it, I don't have the specialized containment facilities 00:05:27.00 you need to study TB, which is a human aerosol pathogen and, besides, he said, TB grows so 00:05:33.05 slowly I'll be dead before you get your first result. 00:05:36.18 And that was in 1991, and I'm happy to say he's still alive, and... and... 00:05:42.17 and quite well. 00:05:45.13 And... so... what he told me... he gave me an insight and he said, look, there are other 00:05:53.19 strains of mycobacteria, there are other species of mycobacteria that are pathogenic in other 00:05:59.01 animals, that are natural pathogens of other animals. 00:06:02.20 And he said, I... 00:06:03.20 I'm pretty sure there are these ones of... of marine life of fish, because I've seen 00:06:09.19 people get them who were fishermen. 00:06:13.19 He had worked at Brown and knew that Portuguese fishermen got these... this disease... mycobacterial 00:06:20.15 disease on their digits and on their soft tissues. 00:06:24.22 And so I went off to the UCSF library and looked at this very classic manual. 00:06:31.13 It's called Bergey's Manual of Systemic Bacteriology, and I... and I found what he was talking about. 00:06:37.16 He was talking about... he was probably talking about a bacterium called Mycobacterium marinum 00:06:43.13 that was thought to be a close relative of human TB, of the human TB bacterium, and it 00:06:50.01 gave fish TB. 00:06:52.24 It turns out that it also infects humans, and this has been known since the 50s, and 00:07:00.02 I can personally attest to it. 00:07:02.18 And it gives humans disease on their extremities, as Stanley already knew, and... and many clinicians, 00:07:08.23 particularly dermatologists and infectious disease clinicians, already know this. 00:07:13.12 But if you look inside that lesion, you'll see a classic granuloma and actually in many 00:07:18.20 cases it can be indistinguishable from the granulomas caused by the human TB bacterium. 00:07:27.03 But, of course, we know that this bacterium also infects fish, and it was first identified 00:07:34.03 to do so in the Philadelphia Aquarium, where in 1926 fish were dying of some mysterious 00:07:42.20 wasting disease, very similar to human TB. 00:07:46.12 And when they tried to culture these fish to see what bacterium they had, why were they 00:07:52.23 dying?, they couldn't culture anything, but when they looked at the fish by histology 00:07:57.24 they could see these classic red snapper bacteria that looked very much like TB. 00:08:04.05 And then Aronson had the bright idea to culture the... to try to do the cultures at a low 00:08:09.21 temperature that was commensurate with... with the low body temperature of the fish, 00:08:14.02 and then he was able to cultivate, he was able to culture Mycobacterium marinum. 00:08:19.08 And, since then, we've had Mycobacterium marinum sequenced at the Sanger Center, and it turns 00:08:26.22 out to be the closest genetic relative of the human TB bacterium, so I guess we also 00:08:32.16 got quite lucky. 00:08:34.00 And it turns out that Mycobacterium marinum also infects zebrafish. 00:08:39.14 Zebrafish are a pet develop... a pet organism of developmental biologists and are a natural 00:08:46.09 host to Mycobacterium marinum. 00:08:48.20 So, I've got for you, here, down below in... in the bottom panel, the... a human TB granuloma 00:08:56.14 stained by hematoxylin and eosin, which will only stain the host cells but not the bacteria, 00:09:01.20 and what you can see is that you've got a nice organized structure, which is cellular, 00:09:07.23 as evidenced by blue nuclei on the edges, but in the center where that arrowhead is, 00:09:14.12 you'll see that the structures become acellular, because it's undergone necrosis, just as we 00:09:21.20 know that human TB granulomas do. 00:09:24.03 But here's, now... let's take a look, now, at the... at a zebrafish granuloma, and in 00:09:30.03 this granuloma you can see... which... which I've stained, here, with a stain that also 00:09:36.22 stains the bacteria, you can see that it looks very similar and it's a nice cellular organized 00:09:43.13 structure and you can see that there are a few bacteria within macrophages, but where 00:09:48.12 the macrophages have necrosed there are tons of bacteria, 00:09:51.12 which is exactly what you would expect. 00:09:55.11 But the great feature of the zebrafish that makes them so enticing to developmental biologists 00:10:01.11 is that they have a prolonged larval phase when they're transparent. 00:10:06.09 And so you can actually watch things happen and people watch developmental processes happen, 00:10:12.24 but... so we asked, well, can we put in bacteria and watch infection happen? 00:10:18.10 And so, they have a cavity that's called the hindbrain ventricle, which is the equivalent 00:10:22.24 of something in our brain, close to our brain called the fourth ventricle, and so we put 00:10:27.09 in some bacteria there -- I've shown it to you with an arrowhead, there -- and what we 00:10:32.03 saw very quickly was that macrophages came, and you'll see the macrophages sort of chasing 00:10:40.14 after the bacterium like a cat after a mouse, and eventually you'll see this mac... mac... 00:10:45.17 macrophage gets it. 00:10:47.12 And there you've got an infected macrophage. 00:10:51.20 You can now follow these infected macrophages out of the cavity -- this is a few days later 00:10:57.12 -- and you can see that it's just moseying along. 00:11:02.04 The bacteria have grown in the macrophage and -- because it's a permissive macrophage 00:11:08.19 for the... for the... for the bacterium -- and there it is. 00:11:13.20 But what was really exciting to us was that you could see, within a few days, 00:11:19.02 a granuloma form. 00:11:20.22 And here you can see that we've got a granuloma that's already formed and what you're going 00:11:25.08 to see, where that white... white arrow is, a new uninfected macrophage is going to come 00:11:32.00 and then it's going to enter the structure. 00:11:34.20 So, watch this. 00:11:37.13 See? 00:11:39.11 There it comes, and it's going to squeeze its way in between and get in there. 00:11:50.21 So, the granuloma is a highly chemotactic structure 00:11:54.21 that is recruiting new macrophages to come to it. 00:11:59.19 And then we could show all this by... by engineering fish that were transgenic, so that they had 00:12:07.06 green florescent macrophages and red fluorescent neutrophils, which is another cell type that 00:12:12.11 is somewhat involved in granulomas, but not as much as macrophages. 00:12:16.17 And what you can... and now we've infected the fish with blue fluorescent bacteria and 00:12:20.10 you can see that the... that we've got a nice tight bona fide epithelioid granuloma with 00:12:28.00 infected macrophages. 00:12:29.13 So, this was good. 00:12:31.10 As we were developing this model, my colleague and friend, David Sherman, 00:12:37.11 made a suggestion to us. 00:12:40.16 He... so, it's... people have been searching for virulence determinants in mycobacterium, 00:12:50.03 and one exciting discovery was that a specialized secretion system called the ESX-1 or RD1 locus, 00:12:58.18 which I've shown in white in that top panel, the white genes in the top panel, 00:13:03.24 were involved in virulence. 00:13:05.16 And this was very exciting because these... it turns out that this was the locus that 00:13:10.09 was missing in the attenuated vaccine strain, BCG, that was made by serial passage in... 00:13:17.14 in the 1920s, and now we finally knew the molecular basis of its attenuation. 00:13:24.19 So, Mycobacterium marinum, not surprisingly, has a locus that looks virtually identical. 00:13:31.19 And David kept telling me, look, make a mutation in this and let's see how it really works, 00:13:38.10 because everyone knows it's attenuated, but a lot of these animal models are black boxes 00:13:42.24 because you only get to see the end result, and he could see that we would be able to 00:13:47.01 get some insights about the actual sequence of what was got... what was different. 00:13:52.04 And, when I was a bit slow to do this, he actually had someone in his lab make the mutation... 00:13:59.04 the mutant for me, and he gave it to us and he said, take this. 00:14:02.02 So, at this point, we were sort of shamed into doing this quickly, and we was 00:14:07.03 Hannah Volkman, who had joined my lab at... as a graduate student, and Hannah showed very quickly 00:14:12.08 that, yes, if you put this mutant into zebrafish larva, it was attenuated. 00:14:17.16 The animals didn't die and if you looked at the bacterial counts you could see that the 00:14:23.03 bacteria didn't grow as well. 00:14:24.03 So, this was good because it showed us that it was behaving just like you would expect. 00:14:28.12 But, here came the surprise. 00:14:30.21 And, at this point... by this point, Hannah had recruited some of her colleagues -- Dana 00:14:36.12 Beery, on the left, who was a technician in the lab, and Hilary Clay, a graduate student 00:14:41.01 who was Hannah's very good friend -- and she got them to join in this... in this quest 00:14:46.01 to see what was going on. 00:14:48.15 And what they found was something quite interesting. 00:14:52.13 Because, if you look at the fish on top, they are infected with wild-type bacterium, and, 00:14:58.19 if you look at the close-up on the top right, 00:15:01.17 you can see that a nice big granuloma has formed. 00:15:04.22 But if you look at the mutant, what you can see is that, even if you inject many, many 00:15:09.22 more bacteria, just to compensate, so that you get more... as many bacteria as with the 00:15:16.10 wild-type, the macrophages pack up with the bacteria, as you see on that bottom-right 00:15:21.09 panel, but there's... they don't form granulomas. 00:15:25.13 Now, this seemed opposite of what you'd expect, because if... if granulomas are good for the 00:15:32.09 host, as what everyone in TB... in the field of TB thought... people have thought that 00:15:38.12 the granuloma is a critical host protective structure that walls off the bacteria and, 00:15:46.00 while it's not always successful in eradicating the bacteria, it sure as heck tries to do 00:15:51.14 so, and is... is... is pretty good at it. 00:15:54.12 Now, if that's the case, then we should see more granuloma formation with that mutant, 00:16:00.03 but we saw less. 00:16:02.05 So, Hannah took a close look at this and she was able to observe fish as the granuloma 00:16:08.24 formed by serial imaging, and what she showed was that, when the granuloma formed, the number 00:16:16.19 of infected macrophages went up dramatically, as did the number of bacteria. 00:16:21.19 So, the granuloma was actually promoting growth rather than restricting growth. 00:16:26.18 So, why might this be? 00:16:28.15 Because here we are saying that a... the... you know, this... this immunological structure 00:16:34.07 that really should be killing the bacteria is actually promoting growth. 00:16:40.24 And the answer to this came of... both from Hannah's work and from a new graduate student 00:16:49.20 who joined, an MD/PhD student, Muse Davis, and what we found was happening was that, 00:16:57.02 when there's an infected macrophage, when new macrophages come to it, for some reason, 00:17:04.13 if they have that ESX1 locus, the bacteria are spreading quickly from macrophage to macrophage. 00:17:12.16 You can see, within 48 hours, you've gone from one infected macrophage in that top-left 00:17:17.12 panel to many infected macrophages, whereas if you didn't have that locus, if the bacterium 00:17:23.24 didn't have that locus, then that one mac... macrophage just remains one great big macrophage. 00:17:29.09 And the bacteria are just growing in it, but obviously they're not doing as well as if 00:17:33.09 they can spread to new macrophages. 00:17:37.20 And so it turned out that the bacterium is 00:17:40.07 using this locus to spread from one macrophage to another. 00:17:44.16 Now, how does this happen? 00:17:46.13 What... what Muse found was that, if the initial macrophage was infected with bacteria that 00:17:54.03 contained this locus, then somehow that macrophage was able to exert a rapidly chemotactic effect 00:18:03.18 on... on macrophages around it, or... or even far away, so that they came. 00:18:10.18 And you can see that the macrophages in the top panel have these protrusions that reflect 00:18:16.15 that they're highly chemotactic and are responding to a chemotactic gradient, and our racing 00:18:22.13 into this structure. 00:18:24.07 In contrast, if that initial macrophage didn't have this locus, then the incoming macrophages 00:18:31.03 are coming in very, very slowly, and they clearly are not experiencing a chemotactic 00:18:36.15 gradient; they don't have that big... great big protrusion. 00:18:40.18 And, even once they get into the granuloma, they behave very differently. 00:18:45.06 The wild-type macrophages move far and wide within the granuloma, and rapidly, whereas 00:18:50.13 the mutant macrophages, the few that do come, are just sort of sitting there 00:18:54.23 like bumps on a log. 00:18:58.20 And this is the kind of movie that gave us that insight that I just talked to you about, 00:19:04.10 that macrophages are used... exploited by the bacteria to spread from cell to cell. 00:19:11.01 So, what happens is that, when a given infected macrophage packs up with... packs up with 00:19:17.08 bacteria, because the bacteria can grow within it, it undergoes an apoptotic death, where 00:19:24.10 it's dead but it's preserved its membranes. 00:19:27.19 And now what happens is this dead cell is recognized by the incoming macrophages, that 00:19:34.22 come and eat it. 00:19:36.05 And I'm going to show you an example, here, of where one... one dead macrophage with the 00:19:42.07 white arrow is going to be engulfed by an incoming macrophage. 00:19:46.19 So, watch this happen. 00:19:48.02 It's... it... you're going to watch it, it's going to come from below, it's kind of like 00:19:52.04 that movie Jaws, where the... you know, the shark comes from below. 00:19:56.06 And, look at it, it's eating it bite by bite. 00:19:59.22 And this is why macrophages might be called what they are -- macrophage for "big eater". 00:20:05.12 And now you've got... this macrophage has been eaten by a new macrophage. 00:20:12.12 But you're going to say, wait a minute, why would this spread the infection? 00:20:15.18 You've gone from one macrophage to another macrophage, so all you've done is conserved 00:20:19.12 the bacteria. 00:20:21.04 But it turns out, when Muse looked closely, that, on average, a given macrophage, given 00:20:26.14 infected, dead macrophage, was eaten, on average, by 2.3 macrophages every 24 hours. 00:20:34.03 And so you can imagine... you can see how the bug is using the macrophage to expand 00:20:39.24 its numbers. 00:20:42.06 And not only that, but we showed that the bacterium also induces the death of the macrophages. 00:20:47.21 Here's a TUNEL stain on the left and this... this death... there are probably many bacterial 00:20:53.03 determinants that do this, but one of them is that self... same locus, ESX1, that also 00:20:58.11 induces the macrophages to come. 00:21:00.06 So, it's got a two-pronged effect. 00:21:02.05 It's inducing death of the infected macrophage and, separately, it's recruiting new macrophages 00:21:07.18 to come to it so that they can engulf the dead macrophage, and... and produce infection. 00:21:15.22 So, to summarize this, let's take a look at what happens in the mutant first. 00:21:23.19 Because, in the mutant, the granuloma might actually be functioning as a host-protective 00:21:29.14 structure, as it might be meant to be. 00:21:34.06 It's you've... you've got an infected macrophage, it dies at a slow rate, macro... new macrophages 00:21:42.23 are slowly recruited at a... at a respectable pace. 00:21:46.10 And, now, they can eat the new macrophage one by one on one, and there's some time for 00:21:53.07 the cells to also kill the dying cell, to also kill the bacteria before they're eaten, 00:21:59.10 so you could imagine there's an attrition of bacterial infection. 00:22:02.19 This might be why the BCG vaccine strain is attenuated. 00:22:06.11 But, paradoxically, instead of sort of thwarting these host-protective processes, as you might 00:22:14.19 imagine a pathogenic bacterium might do, the... the pathogen actually accelerates these processes, 00:22:21.05 so it converts them from being a host-protective to a host-detrimental process. 00:22:26.04 So, simply by speeding up the rate of cell death, and speeding up the recruitment of 00:22:30.19 new macrophages, it's... it's just using the macrophage niche to spread in from cell to 00:22:37.12 cell, and therefore expand itself in the granuloma. 00:22:40.20 And this is quite a nifty thing to do, I think. 00:22:45.04 So... okay. 00:22:46.13 But the question now is, how do... how do... how does this ESX1 locus induce the recruitment 00:22:53.04 of new macrophages? 00:22:54.21 And so, for this part, Hannah was joined by Tamara Pozos, who was a pediatric infectious 00:23:00.13 diseases fellow who joined the lab, and together they did a microarray where they looked at 00:23:05.10 fish that were infected with wild-type or mutant bacteria to identify host genes that 00:23:11.03 might be different between the two. 00:23:13.00 And the gene that stuck out was matrix metalloproteinase 9. 00:23:17.18 And this is an extracellular... an... an enzyme of that... that models... 00:23:23.06 remodels the extracellular matrix. 00:23:25.22 And they were even able to show, not only by transcriptional analyses but also by doing 00:23:31.06 a gelatinase assay on the fish for the actual activity of this enzyme, that MMP9 was induced 00:23:39.20 upon wild-type infection but not upon mutant infection. 00:23:44.16 Now, that's fine. 00:23:47.03 But if MMP9 is... induction of MMP9 is responsible for the ESX-mediated acceleration of macrophage 00:23:57.13 recruitment, then, if you make a MMP9 mutant, that mutant should be attenuated even with 00:24:03.05 wild-type infection. 00:24:04.08 And, sure enough, when they looked they saw that the MMP9 mutant, which is shown on the 00:24:09.17 bottom, there, that fish, was attenuated for infection, and it had very few granulomas. 00:24:17.15 So, it was behaving just like the bacterial mutant and therefore it was the partner. 00:24:24.09 So, so this was nice and then... but then we started to look into what MMP9 does and 00:24:30.03 then it turns out that MMP9 is involved in the pathogenesis of arthritis, and cancers, 00:24:37.14 and other inflammatory conditions, and often in those cases the mac... it's the mac... 00:24:43.07 it's a macrophage that is the bad actor, that is making MMP9 and... and causing trouble 00:24:49.06 in these lesions. 00:24:50.06 So, of course, we thought, well, okay a macrophage gets infected with the bacteria it now induces 00:24:56.17 MMP9 in the macrophage, and now that... that is secreted and calls in new macrophages. 00:25:03.11 But when we did in situ analyses there... to look at where the MMP9 was being made, 00:25:10.13 here is a granuloma, and the MMP9 is labeled green and the macrophage... macrophages are 00:25:17.14 labeled red, and what you can see is that the MMP9 is not in the... in the macrophages 00:25:22.15 of the granuloma. 00:25:23.17 But, rather, it's in the epithelial cells surrounding the granuloma. 00:25:29.13 And so what... what seems to be happening is that an infected macrophage secretes something 00:25:37.18 from that secretion system that goes and talks to the epithelial cell 00:25:43.02 and induces it to make MMP9. 00:25:45.14 And the MMP9 now calls in new macrophages, and this... this... this strategy was called 00:25:52.09 "subversion from the sidelines" by my friend and colleague, Bill Bishai at Hopkins, and 00:25:57.20 I rather like how he put it. 00:26:00.11 So, why might that be? 00:26:03.05 You could imagine that the bacterium wants to tamp down immunity in the infect... in 00:26:10.05 the actual... in the macrophage itself, because it has to survive in there. 00:26:13.21 So, it's doing that and, meanwhile, it's inducing an inflammatory program in a neighboring cell, 00:26:19.24 so that it can bring more macrophages, infect, and then subvert them. 00:26:25.03 Okay. 00:26:26.04 So, but... but... so this is so... so this is how the bacterium uses the innate immune 00:26:33.23 phase of the granuloma to promote its... its growth and expansion. 00:26:40.14 Of course, then the bac... the granuloma matures and other things happen and, as I told you, 00:26:47.01 one of the things that happens is epithelioid transformation, these tight interdigitated 00:26:52.09 projections, that too has been known for, oh, a hundred years or so, and that very reasonably 00:27:00.01 was thought to be a host-protective mechanism that would sort of wall off the bacteria and... 00:27:09.08 and perhaps be... somehow help the host, despite all these strategies of the bacterium. 00:27:14.06 Well, very recently, work from David Tobin's lab, also done in the zebrafish, that this 00:27:20.11 too turns out not to be the case. 00:27:24.10 Mark Cronin and David Tobin have shown that epithelioid transformation of the macrophage 00:27:31.09 is also something that the bacterium is benefiting from. 00:27:34.16 If they inhibit it, then they can... they get less infection than if it's there. 00:27:43.00 And, of course, they're working out the details of how this might be, but what it's telling 00:27:47.02 you is that practically every step that we might predict will help the host can be taken 00:27:52.21 advantage of by the bacterium. 00:27:56.04 So, in my first lecture, I told you that most people actually clear infection and they do 00:28:02.11 so after the adaptive phase of the... of the granuloma has kicked in. 00:28:06.22 So, it's very clear that... that at some point the granuloma can fight back and can... can 00:28:14.21 eradicate or at least suppress infection. 00:28:17.14 And many, many people who specialize in the areas of adaptive immunity and TB are... are 00:28:24.17 working on this. 00:28:26.14 But I want to close by saying that, while the adaptive immunologists may not know as 00:28:32.11 much about this as they want to, and they're working hard to figure it out, the bacterium 00:28:37.01 seems to know quite a little... quite a bit about this... these immune mechanisms, because 00:28:42.11 what these people so... who worked on it so far can tell you is that it tries really hard 00:28:48.08 to delay and inhibit adaptive immunity, so that the adaptive immune elements that come 00:28:54.18 into the granuloma do so late. 00:28:58.10 And this gives time for the mechanisms that I've just been telling you about to help bacteria 00:29:04.20 expand in the innate context. 00:29:07.20 I'll close by thanking the many people whose research I've described to you -- they're 00:29:14.04 both students and from my lab, as well as colleagues and collaborators from outside 00:29:19.19 my lab, and, indeed, from across the world. 00:29:23.08 Thank you.